Bottom Line:
First we describe the effect of mutations on six different positions chosen to destabilize Tdt Loop1 structure, either by alanine substitution or by deletion; they result at most in a reduction of Tdt activity, but adding Co(++) restores most of this Tdt activity.Among them, the single-point mutant F401A displays a sequence-specific replicative polymerase phenotype that is stable upon Co(++) addition.These results are discussed in light of the available crystal structures.

ABSTRACTTerminal deoxynucleotidyltransferase (Tdt) and DNA polymerase mu (pol mu) are two eukaryotic highly similar proteins involved in DNA processing and repair. Despite their high sequence identity, they differ widely in their activity: pol mu has a templated polymerase activity, whereas Tdt has a non-templated one. Loop1, first described when the Tdt structure was solved, has been invoked as the major structural determinant of this difference. Here we describe attempts to transform Tdt into pol mu with the minimal number of mutations in and around Loop1. First we describe the effect of mutations on six different positions chosen to destabilize Tdt Loop1 structure, either by alanine substitution or by deletion; they result at most in a reduction of Tdt activity, but adding Co(++) restores most of this Tdt activity. However, a deletion of the entire Loop1 as in pol lambda does confer a limited template-dependent polymerase behavior to Tdt while a chimera bearing an extended pol mu Loop1 reproduces pol mu behavior. Finally, 16 additional substitutions are reported, targeted at the two so-called 'sequence determinant' regions located just after Loop1 or underneath. Among them, the single-point mutant F401A displays a sequence-specific replicative polymerase phenotype that is stable upon Co(++) addition. These results are discussed in light of the available crystal structures.

Figure 2: Substitution Tdt Mutants in Loop1. (A) Catalytic activity without CoCl2 but in the presence of 4 mM MgCl2 (from left to right) of wild-type Tdt, S383A, T384A and F385A mutants is shown for each dNTP and a mixture of all dNTPs, using the oligonucleotide duplex indicated on top as a primer. (B) The activity without CoCl2 but in the presence of 4 mM MgCl2 (from left to right) of Tdt wild-type, P391L, S392A and D396A mutants is shown for each dNTP and a mixture of all dNTPs, all using the oligonucleotide duplex indicated on top as a primer.

Mentions:
The initial strategy was to destabilize the structure of Loop1, in order to render it more prone to be displaced by the template strand. Mutations were designed to target those residues that were previously described to contribute to the stability of Tdt Loop1 structure (22). First, substitutions by alanine were tried, then cumulative deletions. Both strictly conserved and less highly conserved residues were targeted. Then a larger deletion inspired by pol λ and a chimera with pol μ Loop1 were tried. Finally, the study was further extended to two adjacent regions that are called sequence-determinant regions (SD1 and SD2) located downstream of Loop1 (SD1) and underneath it (SD2): they involve stretches of residues that are strictly conserved in all known Tdt sequences and all known pol μ sequences but differ one from the other [see Figures 1 and 2 of (22)].Figure 2.

Figure 2: Substitution Tdt Mutants in Loop1. (A) Catalytic activity without CoCl2 but in the presence of 4 mM MgCl2 (from left to right) of wild-type Tdt, S383A, T384A and F385A mutants is shown for each dNTP and a mixture of all dNTPs, using the oligonucleotide duplex indicated on top as a primer. (B) The activity without CoCl2 but in the presence of 4 mM MgCl2 (from left to right) of Tdt wild-type, P391L, S392A and D396A mutants is shown for each dNTP and a mixture of all dNTPs, all using the oligonucleotide duplex indicated on top as a primer.

Mentions:
The initial strategy was to destabilize the structure of Loop1, in order to render it more prone to be displaced by the template strand. Mutations were designed to target those residues that were previously described to contribute to the stability of Tdt Loop1 structure (22). First, substitutions by alanine were tried, then cumulative deletions. Both strictly conserved and less highly conserved residues were targeted. Then a larger deletion inspired by pol λ and a chimera with pol μ Loop1 were tried. Finally, the study was further extended to two adjacent regions that are called sequence-determinant regions (SD1 and SD2) located downstream of Loop1 (SD1) and underneath it (SD2): they involve stretches of residues that are strictly conserved in all known Tdt sequences and all known pol μ sequences but differ one from the other [see Figures 1 and 2 of (22)].Figure 2.

Bottom Line:
First we describe the effect of mutations on six different positions chosen to destabilize Tdt Loop1 structure, either by alanine substitution or by deletion; they result at most in a reduction of Tdt activity, but adding Co(++) restores most of this Tdt activity.Among them, the single-point mutant F401A displays a sequence-specific replicative polymerase phenotype that is stable upon Co(++) addition.These results are discussed in light of the available crystal structures.

ABSTRACTTerminal deoxynucleotidyltransferase (Tdt) and DNA polymerase mu (pol mu) are two eukaryotic highly similar proteins involved in DNA processing and repair. Despite their high sequence identity, they differ widely in their activity: pol mu has a templated polymerase activity, whereas Tdt has a non-templated one. Loop1, first described when the Tdt structure was solved, has been invoked as the major structural determinant of this difference. Here we describe attempts to transform Tdt into pol mu with the minimal number of mutations in and around Loop1. First we describe the effect of mutations on six different positions chosen to destabilize Tdt Loop1 structure, either by alanine substitution or by deletion; they result at most in a reduction of Tdt activity, but adding Co(++) restores most of this Tdt activity. However, a deletion of the entire Loop1 as in pol lambda does confer a limited template-dependent polymerase behavior to Tdt while a chimera bearing an extended pol mu Loop1 reproduces pol mu behavior. Finally, 16 additional substitutions are reported, targeted at the two so-called 'sequence determinant' regions located just after Loop1 or underneath. Among them, the single-point mutant F401A displays a sequence-specific replicative polymerase phenotype that is stable upon Co(++) addition. These results are discussed in light of the available crystal structures.